Neurofibromatosis type 2 (NF-2) is a dominantly inherited genetic disorder that results from variants in the tumor suppressor gene, neurofibromin 2 (NF2). Here, we report the generation of a conditional zebrafish model of neurofibromatosis established by inducible genetic knockout of nf2a/b, the zebrafish homologs of human NF2. Analysis of nf2a and nf2b expression revealed ubiquitous expression of nf2b in the early embryo, with overlapping expression in the neural crest and its derivatives and in the cranial mesenchyme. In contrast, nf2a displayed lower expression levels. Induction of nf2a/b knockout at early stages increased the proliferation of larval Schwann cells and meningeal fibroblasts. Subsequently, in adult zebrafish, nf2a/b knockout triggered the development of a spectrum of tumors, including vestibular Schwannomas, spinal Schwannomas, meningiomas and retinal hamartomas, mirroring the tumor manifestations observed in patients with NF-2. Collectively, these findings highlight the generation of a novel zebrafish model that mimics the complexities of the human NF-2 disorder. Consequently, this model holds significant potential for facilitating therapeutic screening and elucidating key driver genes implicated in NF-2 onset.
- Klíčová slova
- Cancer, Inducible knockout, Meninges, Neurofibromatosis type 2, Schwann cells, Zebrafish,
- MeSH
- dánio pruhované * genetika embryologie MeSH
- geneticky modifikovaná zvířata MeSH
- genový knockout * MeSH
- larva metabolismus MeSH
- lidé MeSH
- modely nemocí na zvířatech * MeSH
- neurofibromatóza 2 genetika patologie metabolismus MeSH
- neurofibromatózy genetika patologie metabolismus MeSH
- neurofibromin 2 * genetika metabolismus nedostatek MeSH
- proliferace buněk MeSH
- proteiny dánia pruhovaného * genetika metabolismus nedostatek MeSH
- Schwannovy buňky metabolismus patologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- neurofibromin 2 * MeSH
- proteiny dánia pruhovaného * MeSH
Insect prophenoloxidases (PPOs) are important immunity proteins for defending against the invading pathogens and parasites. As a Type-Ⅲ copper-containing proteins, unlike Homo sapiens tyrosinases, the insect PPOs and most bacterial tyrosinases contain no signal peptides for unknown reason, however they can still be released. To this end, we fused different signal peptides to Drosophila melanogaster PPOs for in vitro and in vivo expression, respectively. We demonstrate that an artificial signal peptide can help PPO secretion in vitro. The secreted PPO appeared larger than wild-type PPO on molecular weight sizes due to glycosylation when expressed in S2 cells. Two asparagine residues for potential glycosylation in PPO1 were identified when a signal peptide was fused. After purification, the glycosylated PPO1 lost zymogen activity. When PPO1 containing a signal peptide was over-expressed in Drosophila larvae, the glycosylation and secretion of PPO1 was detected in vivo. Unlike insect PPO, human tyrosinase needs a signal peptide for protein expression and maintaining enzyme activity. An artificial signal peptide fused to bacterial tyrosinase had no influence on the protein expression and enzyme activity. These Type-Ⅲ copper-containing proteins from different organisms may evolve to perform their specific functions. Intriguingly, our study revealed that the addition of calcium inhibits PPO secretion from the transiently cultured larval hindguts in vitro, indicating that the calcium concentration may regulate PPO secretion. Taken together, insect PPOs can maintain enzyme activities without any signal peptide.
- Klíčová slova
- Enzyme activity, Glycosylation, Insect, Prophenoloxidase, Signal peptide,
- MeSH
- buněčné linie MeSH
- Drosophila melanogaster * imunologie metabolismus MeSH
- glykosylace MeSH
- hmyzí proteiny metabolismus genetika MeSH
- katecholoxidasa * metabolismus MeSH
- larva metabolismus MeSH
- lidé MeSH
- prekurzory enzymů * metabolismus MeSH
- proteinové prekurzory metabolismus MeSH
- proteiny - lokalizační signály * MeSH
- proteiny Drosophily metabolismus genetika MeSH
- tyrosinasa metabolismus MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- hmyzí proteiny MeSH
- katecholoxidasa * MeSH
- prekurzory enzymů * MeSH
- pro-phenoloxidase MeSH Prohlížeč
- proteinové prekurzory MeSH
- proteiny - lokalizační signály * MeSH
- proteiny Drosophily MeSH
- tyrosinasa MeSH
- vápník MeSH
Emerging environmental contaminants, organophosphate flame retardants (OPFRs), pose significant threats to ecosystems and human health. Despite numerous studies reporting the toxic effects of OPFRs, research on their epigenetic alterations remains limited. In this study, we investigated the effects of exposure to 2-ethylhexyl diphenyl phosphate (EHDPP), tricresyl phosphate (TMPP), and triphenyl phosphate (TPHP) on DNA methylation patterns during zebrafish embryonic development. We assessed general toxicity and morphological changes, measured global DNA methylation and hydroxymethylation levels, and evaluated DNA methyltransferase (DNMT) enzyme activity, as well as mRNA expression of DNMTs and ten-eleven translocation (TET) methylcytosine dioxygenase genes. Additionally, we analyzed genome-wide methylation patterns in zebrafish larvae using reduced-representation bisulfite sequencing. Our morphological assessment revealed no general toxicity, but a statistically significant yet subtle decrease in body length following exposure to TMPP and EHDPP, along with a reduction in head height after TPHP exposure, was observed. Eye diameter and head width were unaffected by any of the OPFRs. There were no significant changes in global DNA methylation levels in any exposure group, and TMPP showed no clear effect on DNMT expression. However, EHDPP significantly decreased only DNMT1 expression, while TPHP exposure reduced the expression of several DNMT orthologues and TETs in zebrafish larvae, leading to genome-wide aberrant DNA methylation. Differential methylation occurred primarily in introns (43%) and intergenic regions (37%), with 9% and 10% occurring in exons and promoter regions, respectively. Pathway enrichment analysis of differentially methylated region-associated genes indicated that TPHP exposure enhanced several biological and molecular functions corresponding to metabolism and neurological development. KEGG enrichment analysis further revealed TPHP-mediated potential effects on several signaling pathways including TGFβ, cytokine, and insulin signaling. This study identifies specific changes in DNA methylation in zebrafish larvae after TPHP exposure and brings novel insights into the epigenetic mode of action of TPHP.
- MeSH
- dánio pruhované * genetika růst a vývoj metabolismus MeSH
- larva * účinky léků genetika metabolismus růst a vývoj MeSH
- metylace DNA * účinky léků MeSH
- organofosfáty * toxicita MeSH
- retardanty hoření toxicita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- organofosfáty * MeSH
- retardanty hoření MeSH
- triphenyl phosphate MeSH Prohlížeč
The larvae of the moth Hyalophora cecropia spin silk cocoons with morphologically distinct layers. We investigated the expression of the individual silk protein components of these cocoons in relation to the morphology of the silk gland and its affiliation to the different layers of the cocoon. The study used transcriptomic and proteomic analyses to identify 91 proteins associated with the silk cocoons, 63 of which have a signal peptide indicating their secretory nature. We checked the specificity of their expression in different parts of the SG and the presence of the corresponding protein products in each cocoon layer. Differences were observed among less abundant proteins with unclear functions. The representation of proteins in the inner envelope and intermediate space was similar, except for a higher proportion of probable contaminating proteins, mostly originating from the gut. On the other hand, the outer envelope contains a number of putative enzymes with unclear function. However, the protein most specific to the outer layer has sequence homology to putative serine/threonine kinase-like proteins and some adhesive proteins, and its closest homolog in Bombyx mori was found in the scaffold silk. This research provides valuable insights into the silk production of the cecropia moth, highlighting both similarities and differences to other moth species.
- Klíčová slova
- Fibrohexamerin-like protein, Lepidoptera, Silk, Silk antibacterial proteins, Silk kinase, Silk laccase,
- MeSH
- hedvábí * metabolismus genetika MeSH
- hmyzí proteiny * metabolismus genetika MeSH
- larva metabolismus genetika růst a vývoj MeSH
- můry * genetika metabolismus MeSH
- proteomika MeSH
- stanovení celkové genové exprese MeSH
- transkriptom MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- hedvábí * MeSH
- hmyzí proteiny * MeSH
Activation of immune cells requires the remodeling of cell metabolism in order to support immune function. We study these metabolic changes through the infection of Drosophila larvae by parasitoid wasp. The parasitoid egg is neutralized by differentiating lamellocytes, which encapsulate the egg. A melanization cascade is initiated, producing toxic molecules to destroy the egg while the capsule also protects the host from the toxic reaction. We combined transcriptomics and metabolomics, including 13C-labeled glucose and trehalose tracing, as well as genetic manipulation of sugar metabolism to study changes in metabolism, specifically in Drosophila hemocytes. We found that hemocytes increase the expression of several carbohydrate transporters and accordingly uptake more sugar during infection. These carbohydrates are metabolized by increased glycolysis, associated with lactate production, and cyclic pentose phosphate pathway (PPP), in which glucose-6-phosphate is re-oxidized to maximize NADPH yield. Oxidative PPP is required for lamellocyte differentiation and resistance, as is systemic trehalose metabolism. In addition, fully differentiated lamellocytes use a cytoplasmic form of trehalase to cleave trehalose to glucose and fuel cyclic PPP. Intracellular trehalose metabolism is not required for lamellocyte differentiation, but its down-regulation elevates levels of reactive oxygen species, associated with increased resistance and reduced fitness. Our results suggest that sugar metabolism, and specifically cyclic PPP, within immune cells is important not only to fight infection but also to protect the host from its own immune response and for ensuring fitness of the survivor.
- MeSH
- buněčná diferenciace MeSH
- Drosophila melanogaster metabolismus parazitologie MeSH
- Drosophila metabolismus parazitologie MeSH
- glukosa * metabolismus MeSH
- glykolýza MeSH
- hemocyty * metabolismus MeSH
- interakce hostitele a parazita MeSH
- larva metabolismus parazitologie MeSH
- odolnost vůči nemocem MeSH
- pentózofosfátový cyklus * MeSH
- sršňovití metabolismus fyziologie MeSH
- trehalosa * metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The metabolically demanding nature of immune response requires nutrients to be preferentially directed towards the immune system at the expense of peripheral tissues. We study the mechanisms by which this metabolic reprograming occurs using the parasitoid infection of Drosophila larvae. To overcome such an immune challenge hemocytes differentiate into lamellocytes, which encapsulate and melanize the parasitoid egg. Hemocytes acquire the energy for this process by expressing JAK/STAT ligands upd2 and upd3, which activates JAK/STAT signaling in muscles and redirects carbohydrates away from muscles in favor of immune cells. METHODS: Immune response of Drosophila larvae was induced by parasitoid wasp infestation. Carbohydrate levels, larval locomotion and gene expression of key proteins were compared between control and infected animals. Efficacy of lamellocyte production and resistance to wasp infection was observed for RNAi and mutant animals. RESULTS: Absence of upd/JAK/STAT signaling leads to an impaired immune response and increased mortality. We demonstrate how JAK/STAT signaling in muscles leads to suppression of insulin signaling through activation of ImpL2, the inhibitor of Drosophila insulin like peptides. CONCLUSIONS: Our findings reveal cross-talk between immune cells and muscles mediates a metabolic shift, redirecting carbohydrates towards immune cells. We emphasize the crucial function of muscles during immune response and show the benefits of insulin resistance as an adaptive mechanism that is necessary for survival.
- Klíčová slova
- Drosophila, Immunity, Insulin resistance, JAK/STAT signaling, Metabolism,
- MeSH
- Drosophila genetika MeSH
- imunita MeSH
- inzulinová rezistence * MeSH
- Janus kinasy metabolismus MeSH
- larva metabolismus MeSH
- proteiny Drosophily * metabolismus MeSH
- proteiny vázající insulinu podobné růstové faktory metabolismus MeSH
- sacharidy MeSH
- sršňovití * metabolismus MeSH
- svaly MeSH
- transkripční faktory STAT metabolismus MeSH
- transkripční faktory metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- ImpL2 protein, Drosophila MeSH Prohlížeč
- Janus kinasy MeSH
- proteiny Drosophily * MeSH
- proteiny vázající insulinu podobné růstové faktory MeSH
- sacharidy MeSH
- transkripční faktory STAT MeSH
- transkripční faktory MeSH
The uptake of insecticidal Cry1Ab from genetically engineered (GE) maize, via herbivore Rhopalosiphum padi, to a predator Harmonia axyridis and its potential intergenerational transfer were investigated. Cry1Ab concentration was found to be 400-fold lower in R. padi compared to GE maize, and more than two-fold lower in H. axyridis. For 62% of H. axyridis samples, Cry1Ab was under the limit of detection (LOD), for another 13% were under the limit of quantification (LOQ). The concentration of Cry1Ab was similar between H. axyridis exposed short-term and long-term with the exception of adults after long-term. There was no correlation between Cry1Ab in females and eggs and neonates. The performance of H. axyridis was comparable between Cry1Ab and control. Histological investigation did not show any pathological changes in the digestive and reproductive systems. The detected route of exposure is unlikely to be important for functional biological control by H. axyridis in agroecosystem.
- Klíčová slova
- Bt maize, Cry protein, Harmonia axyridis, Toxin accumulation, Toxin uptake,
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- brouci * metabolismus MeSH
- endotoxiny * MeSH
- geneticky modifikované rostliny metabolismus MeSH
- hemolyziny genetika metabolismus MeSH
- kukuřice setá genetika metabolismus MeSH
- larva metabolismus MeSH
- lidé MeSH
- novorozenec MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bakteriální proteiny MeSH
- endotoxiny * MeSH
- hemolyziny MeSH
Most multicellular organisms are freeze sensitive, but the ability to survive freezing of the extracellular fluids evolved in several vertebrate ectotherms, some plants, and many insects. Here, we test the coupled hypotheses that are perpetuated in the literature: that irreversible denaturation of proteins and loss of biological membrane integrity are two ultimate molecular mechanisms of freezing injury in freeze-sensitive insects and that seasonally accumulated small cryoprotective molecules (CPs) stabilize proteins and membranes against injury in freeze-tolerant insects. Using the drosophilid fly, Chymomyza costata, we show that seven different soluble enzymes exhibit no or only partial loss of activity upon lethal freezing stress applied in vivo to whole freeze-sensitive larvae. In contrast, the enzymes lost activity when extracted and frozen in vitro in a diluted buffer solution. This loss of activity was fully prevented by adding low concentrations of a wide array of different compounds to the buffer, including C. costata native CPs, other metabolites, bovine serum albumin (BSA), and even the biologically inert artificial compounds HistoDenz and Ficoll. Next, we show that fat body plasma membranes lose integrity when frozen in vivo in freeze-sensitive but not in freeze-tolerant larvae. Freezing fat body cells in vitro, however, resulted in loss of membrane integrity in both freeze-sensitive and freeze-tolerant larvae. Different additives showed widely different capacities to protect membrane integrity when added to in vitro freezing media. A complete rescue of membrane integrity in freeze-tolerant larvae was observed with a mixture of proline, trehalose, and BSA.
- Klíčová slova
- biological membrane integrity, cryopreservation, enzyme activity, freeze tolerance, protein stabilization,
- MeSH
- aklimatizace MeSH
- buněčná membrána metabolismus MeSH
- Ficoll MeSH
- hmyz metabolismus MeSH
- kryoprotektivní látky farmakologie MeSH
- larva metabolismus MeSH
- prolin metabolismus MeSH
- sérový albumin hovězí * MeSH
- trehalosa * MeSH
- zmrazování MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- Ficoll MeSH
- kryoprotektivní látky MeSH
- prolin MeSH
- sérový albumin hovězí * MeSH
- trehalosa * MeSH
Wastewater treatment plant effluents have been identified as a major contributor to increasing anthropogenic pollution in aquatic environments worldwide. Yet, little is known about the potentially adverse effects of wastewater treatment plant effluent on aquatic invertebrates. In this study, we assessed effects of wastewater effluent on the behaviour and metabolic profiles of damselfly larvae (Coenagrion hastulatum), a common aquatic invertebrate species. Four key behavioural traits: activity, boldness, escape response, and foraging (traits all linked tightly to individual fitness) were studied in larvae before and after one week of exposure to a range of effluent dilutions (0, 50, 75, 100%). Effluent exposure reduced activity and foraging, but generated faster escape response. Metabolomic analyses via targeted and non-targeted mass spectrometry methods revealed that exposure caused significant changes to 14 individual compounds (4 amino acids, 3 carnitines, 3 lysolipids, 1 peptide, 2 sugar acids, 1 sugar). Taken together, these compound changes indicate an increase in protein metabolism and oxidative stress. Our findings illustrate that wastewater effluent can affect both behavioural and physiological traits of aquatic invertebrates, and as such might pose an even greater threat to aquatic ecosystems than previously assumed. More long-term studies are now needed evaluate if these changes are linked to adverse effects on fitness. The combination of behavioural and metabolomic assessments provide a promising tool for detecting effects of wastewater effluent, on multiple biological levels of organisation, in aquatic ecosystems.
- MeSH
- bezobratlí metabolismus MeSH
- chemické látky znečišťující vodu * metabolismus MeSH
- ekosystém MeSH
- larva metabolismus MeSH
- odpadní voda * chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chemické látky znečišťující vodu * MeSH
- odpadní voda * MeSH
Triatomine bugs are the blood feeding insect vectors transmitting Chagas disease to humans, a neglected tropical disease that affects over 8 million people, mainly in Latin America. The behavioral responses to host cues and bug signals in Rhodnius prolixus are state dependent, i.e., they vary as a function of post-ecdysis age. At the molecular level, these changes in behavior are probably due to a modulation of peripheral and central processes. In the present study, we report a significant modulation of the expression of a large set of sensory-related genes. Results were generated by means of antennal transcriptomes of 5th instar larvae along the first week (days 0, 2, 4, 6 and 8) after ecdysis sequenced using the Illumina HiSeq platform. Significant age-induced changes in transcript abundance were established for more than 6120 genes (54,7% of 11,186 genes expressed) in the antenna of R. prolixus. This was especially true between the first two days after ecdysis when more than 2500 genes had their expression significantly altered. In contrast, expression profiles were almost identical between day 6 and 8, with only a few genes showing significant modulation of their expression. A total of 86 sensory receptors, odorant carriers and odorant degrading enzymes were significantly modulated across age points and clustered into three distinct expression profiles. The set of sensory genes whose expression increased with age (profile 3) may include candidates underlying the increased responsiveness to host cues shown by R. prolixus during the first days after molting. For the first time, we describe the maturation process undergone at the molecular level by the peripheral sensory system of a hemimetabolous insect.
- Klíčová slova
- Age, Antennae, Rhodnius prolixus, Sensory genes, Transcriptome,
- MeSH
- Chagasova nemoc přenos MeSH
- čich genetika MeSH
- hmyz - vektory genetika metabolismus MeSH
- hmyzí geny * MeSH
- larva genetika metabolismus MeSH
- odoranty MeSH
- receptory pachové genetika metabolismus MeSH
- Rhodnius * genetika metabolismus MeSH
- smyslové orgány * embryologie fyziologie MeSH
- stanovení celkové genové exprese MeSH
- transkriptom MeSH
- tykadla členovců * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- receptory pachové MeSH
